|Publication number||US20090261243 A1|
|Application number||US 12/424,817|
|Publication date||Oct 22, 2009|
|Filing date||Apr 16, 2009|
|Priority date||Apr 16, 2008|
|Also published as||EP2110845A1, EP2110845B1, US8274045|
|Publication number||12424817, 424817, US 2009/0261243 A1, US 2009/261243 A1, US 20090261243 A1, US 20090261243A1, US 2009261243 A1, US 2009261243A1, US-A1-20090261243, US-A1-2009261243, US2009/0261243A1, US2009/261243A1, US20090261243 A1, US20090261243A1, US2009261243 A1, US2009261243A1|
|Inventors||Casimir Bamberger, Andreas Bamberger|
|Original Assignee||Casimir Bamberger, Andreas Bamberger|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (4), Referenced by (14), Classifications (9)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The present invention relates to imaging mass spectroscopy and more particular to a method, an apparatus and a computer product program for mass spectroscopy by a time of flight principle.
In general, mass spectrometry allows the identification and quantification of atoms and molecules (hereinafter: “molecules”) that can be ionized. Mass spectrometry is commonly used to characterize the molecular composition of a surface. It is a powerful method to detect, identify, and quantify molecules of different masses. Additional spectra obtained after fragmentation of a molecule can be performed for unambiguous identification of most molecules.
For certain applications like the characterization of surfaces in material sciences or the diagnostics of diseases like cancer, an image with a spatial resolution of the mass spectrum is required.
There are several different methods known to generate a maximum number of ions during each measurement cycle, a prerequisite for fast image acquisition:
For matrix assisted LASER desorption ionization (MALDI) (see: A. F. Maarten Altenaar, PhD-thesis, University Utrecht, Netherlands, 2007), a LASER beam is focused on one spot of the surface analyzed. In most cases, a matrix consisting of an organic compound with high light absorption embeds the molecules to be investigated. A focused laser beam ionizes a small portion of the surface area and a large number of ions are generated by a charge transfer from the ionized matrix to the bio-molecules. The exact mass for each of the resulting ionized molecules is determined by mass spectrometry in a subsequent step, typically in a time of flight mass analyzer. The lateral resolution achieved today comprises about 5 to 10 μm reflecting the maximal focussing of the LASER beam. In rare cases, a resolution of 1 μm can be achieved.
Other methods include fast atom bombardment (FAB). FAB destructively induces the ionization at the point to which the ion beam is focused on the surface. The ion gun generates a fast ion beam that consists for example of In+ or Ga+ ions. As soon as the accelerated ions impact on the target surface, a multitude of small, ionized fragments are generated from the bio-molecules present at this spot. Although FAB massively decomposes the surface of the sample, the ion beam can be focussed to a spot of less than 1 μm in diameter. One of the disadvantages of this technique relates to the strong impact of the ions on the surface which causes a substantial fragmentation of molecules, especially bio-molecules. A variation of FAB employs a liquid metal ion source. For example Bismuth ions can be used as ionization source.
Alternatively, the surface can be bombarded with Fullerenes (C60) that dissipate the kinetic energy upon impact on the surface and therefore lead to a softer ionization. Thus, less fragmentation of the ions generated is caused and therefore larger molecules may be ionized. This technique can be utilized to generate ions from a sub-micrometer spot on a sample.
Subsequently the ionized molecules are separated according to their specific m/z value. Here, m designates the mass and z designates the electric charge of the ion. The standard mass analyzers use either the time of flight (TOF) or quadrupole (Q) mass selection principle. Alternatively, ions may be trapped in an ion trap and their mass determined when expelled from the ion trap during a frequency scan.
Also, ions may be introduced into a cyclotron and their mass is determined based on their resonance frequency in a frequency scan. The m/z value of a molecule is correlated to the resonance frequency and determined by a Fourier transformation of the frequency spectra measured. This non-destructive principle of mass determination, also referred to as Fourier Transformation-Ion Cyclotron Resonance (FT-ICR), provides very high mass accuracy.
The mass selection principles outlined above, or any homo- or hetero-mer combination of these methods, are conventionally known. For an un-ambiguous identification of a molecule, secondary ions are generated in a collision cell located between two mass selecting units. For example, in a TOF/TOF setup secondary ions are generated in a collision cell.
An already well-known technology to reconstruct mass spectrometric images of a two dimensional surface is based on a single spot analysis (scanning principle). The ionization is induced in a small region (hereinafter: “spot”) on the sample surface. A typical spot size is typically around 10 μm to 100 μm in diameter. The ions generated in the spot are detected in a mass spectrometer, e.g. a standard time of flight mass spectrometer. This allows determining not only the exact mass but also the abundance of each specific ion within the spot.
The mass spectrometer acquires mass spectra of adjacent spots and thereby scans the surface. Following data acquisition, a four-dimensional map or “picture” is assembled in which the x- and y-axis reflect the imaged surface and the z-axis the mass spectrum. The fourth dimension represents the ion rate measured. An image reflecting the ion abundance distribution of one m/z value over the surface can then be easily visualized.
When mass spectrometric images are reconstructed based on the scanning principle, the mass spectrometer scans the surface along a predetermined path and with a defined step-width. Typically, the instrument acquires more than a hundred and up to one thousand spectra at each individual spot. First, the mass spectrum from each point on a surface is recorded separately. Based on this information, the mass spectrometric image is reconstructed from the acquired mass spectra of each individual spot by a point by point reconstruction. Because a high number of repeated measurements at one spot are necessary and the mass spectra are acquired in each spot individually, the process of data acquisition is very time-consuming. In a variation of it, the long data acquisition time is reduced. In this case, the mass spectrum is only acquired at pre-determined spots on the surface and later an extrapolation allows to determine a subset of area to which this particular spectrum might fit additionally.
A start time of the measurement is set in S203, e.g. by detecting the laser pulse used for ionization. A spot of the sample at the predetermined location (X,Y)n is ionized by the ionization beam pulse in step S204. In step S205, the generated ions drift towards the detector and may generate an amplified signal that impinges the detector. In the detector, the number of events per Time of Arrival ToAn (X,Y)n is counted (step S206), where the Time of Arrival is measured relative to the start time. If enough measurements have not been performed for a predetermined location yet to obtain a sufficient statistics, it is decided in S207, to return to S203. Otherwise, it is checked in step 208, if all locations of interest of the sample were investigated.
If this is not the case, a next location for an investigation is determined, S209, and the procedure returns to step S202.
If all locations have been scanned (“yes” in step S208), the measurement part of a duty cycle is finished and a 4-dimensional image may be reconstructed from all cycles of all locations (S210). For that, the data may be arranged in sets of substantially the same Time of Arrival [ToA], representing a certain m/z ratio, location (X,Y)n, and the number of events. If the number of measurement cycles was different at different locations, the number of events at a location should be normalized to the number of cycles at this location. Finally, in step S211 the data may be evaluated and presented. The duty cycle is finished in S212.
In order to reconstruct an image of an extended surface with one of the above schematically described instruments, a scanning process is required. Moreover, determined through the principle of measurement (maximal number of ions per duty cycle), the duty cycles for each measurement are relatively long. Typically, the duty cycle for the determination of a full mass spectrum image requires the number of ionization shots needed to acquire a spectrum with a given number of detected ions multiplied by the number of spots investigated to reconstruct the mass spectrum image.
The mass spectrometers of e.g. the TRIFT series are another attempt to provide a spatially resolved mass spectroscopic image of a surface (A. F. Maarten Altenaar, PhD-thesis, University Utrecht, Netherlands, 2007). The mass spectrometer of the TRIFT series is described here as an example for a mass spectrometer which is used in quality control during semiconductor microcircuit production, or in the investigation of surfaces of biological samples as described in the publication mentioned above.
In such a spectrometer, a time of flight mass separator is used to acquire the two-dimensional image for a very narrow range of m/z values. The time of flight mass separator is constructed such that it provides directional and velocity focussing properties (double focussing), that enable the arrival of a focussed ion image for a selected m/z value at the detector. In this instrument setup three electrostatic field sectors (for example Matzuda plates) are arranged at a 90° angle to each other along the flight path of the ions providing double velocity focussing.
The TRIFT series of time of flight mass spectrometers can image a surface. It can be operated either in a stigmatic or in an astigmatic mode. In the stigmatic mode, the spatial relationship of the ions is preserved until arrival at the detector. A large amount of molecules are ionized on a restricted surface area either with an ion beam or a laser beam illuminating the surface. The ionized molecules are accelerated. One m/z target value (or a narrow m/z target range) is selected during the drift phase by two blankers included in the instrumentation that enable the selection of a narrow m/z window that is observed at the detector. The position sensitive detector records the spatial distribution of the ions within the selected mass range. In detail, after signal conversion and amplification through a microchannel plate, a phosphor imaging screen converts electrons to light that is detected by a CCD camera.
Whereas this instrument images a surface area with spatial resolution, it is limited in its capacity and speed by at least three factors:
First, the instrument is only capable of generating three-dimensional data and not four-dimensional datasets. For each measurement cycle, a narrow m/z range of interest has to be selected, for which a mass spectrometric image is acquired. Therefore, all ions of different m/z values than the selected window are lost during each duty cycle.
Typically, the duty cycle for the determination of a full mass spectrum image requires the number of ionization shots needed to acquire a spectrum with a given number of detected ions multiplied by the number of m/z ranges investigated to reconstruct the mass spectrum image.
Second, the ion signal is converted in an electron signal which induces a light signal at a phosphor-screen which is finally detected by a CCD camera. Although this is a standard procedure for acquiring images of signals converted to a light pulse, it restricts the efficiency of image acquisition by this three step conversion.
Third, the image acquisition requires several milliseconds due to the method used to record the light signal. The read out process of the CCD camera takes much more time than the duty cycle of the whole instrument. The long duty cycle is due to the photo sensor array used in CCD cameras which finally restricts the rate of image acquisition because sample ionization and mass separation within a time of flight mass spectrometer is achieved within microseconds.
In another attempt to imaging mass spectrometry a three layer delay-line anode was used (O. Jagutzki, V. Mergel, K. Ullmann-Pfleger, L. Spielberger, U. Spillmann, R. Dörner, H. Schmidt-Böcking: A broad-application microchannel-plate detector system for advanced particle or photon detection tasks: large area imaging, precise multi-hit timing information and high detection rate, Nucl. Instr. and Meth. in Phys. Res. A, 477 (2002) 244-249). It comprises three individual delay chains. From the relative delay of the signals arriving at the two ends of each delay chain, the position of a single event on that delay line can be obtained. With two independent delay lines, the determination of the location in a detection plane is not unambiguous, if two events occur at the same time at different locations. A third delay line allows for unambiguous identification even in this case of two simultaneous events but still the detector may suffer from ambiguities in the determination of an impact position at higher intensities.
Therefore it is an object of the present invention to overcome the drawbacks of the prior art.
According to aspects of the present invention a method of mass spectrometry determining at the same time the m/z value of a molecule is provided, where the m/z range is not restricted to a small m/z range, and the location of the molecule on the investigated surface is determined simultaneously, thus reducing significantly the time required for recording a mass spectroscopic image.
Such a method according to aspects of the present invention allows a rapid construction of images containing information about the molecular composition of two-dimensional surfaces. The method is comparable to that of a digital camera where the colours are replaced by the time of flight information representing the different types of ions.
According to a first aspect of the present invention, this object is solved by providing a method, setting a start time; extracting ions from a sample by an ionization pulse at a fixed time relative to the start time; accelerating said ions towards a signal generator located at a distance to the sample whereby the distribution of the ions on the sample is isomorphously imaged to the signal generator; generating from each ion, by the signal generator, a signal indicative of the position of the impingement of the ion onto the signal generator; detecting, by a detection element of a detector comprising two laterally separated detection elements, if a part of said signal with at least a pre-defined intensity is received by said detection element; and measuring, by at least one detection element of the detector, a time of arrival relative to the start time when said part of said signal with at least a pre-defined intensity is received by said detection element.
The method according to the first aspect can be a method of imaging mass spectroscopy.
According to the first aspect, the time of flight information of an ion and the position of the signal corresponding to a location of the ion on the sample can be obtained simultaneously, hence avoiding the need for any scanning. E.g., the position of the signal can be obtained by determining the center of gravity of detection elements detecting parts of the signal.
Preferably, this aspect of the invention comprises further measuring, by a detection element of the detector, the intensity of the received part of the signal.
Thus, the position of the signal on the detector can be obtained more precisely. E.g., the center of gravity of the detection elements detecting the signal can be determined from the positions of these detection elements weighted with the measured intensity.
Preferably, this aspect of the invention comprises further performing the ionization by irradiation with a laser beam or with an ion beam that illuminates or impinges on the sample area homogenously.
Thus the ionization can be adapted to the area under investigation and the type of ions and the rate of ions does not depend on the orientation of the sample relative to the ionization means.
More preferably, this aspect of the invention comprises further setting the intensity of ionization to extract an average of ions per detecting element by one pulse without resulting in a signal saturation of the detection element.
In embodiments according to this more preferred aspect, the intensity may be sufficient to generate quickly a mass spectrometric image but not over-saturating the detector. Therefore, the abundance and distribution of ions can be easier determined.
Preferably, the isomorphous imaging of this aspect of the invention comprises further a diminishment or an enlargement.
A diminishment can be obtained by bundling ions from a large surface area, an enlargement would allow microscopic imaging of the sample surface.
Preferably the ion trajectories between the sample and the signal generator are straight, bend, in a closed loop, single or multiple time reflected before arrival to the signal generator.
E.g., the ion trajectories can follow closed loops or are bend to more than 90° change in direction and thereby reflected. More preferably, ions are reflected in a main axis symmetric assembly of two ion mirrors with one or more lenses between two ion mirrors.
Preferably, this aspect of the invention comprises further data processing methods during or after data acquisition that allow reconstructing the position of the signal from the positions of the detection elements detecting a part of the signal.
More preferably, the detector according to this aspect of the invention comprises at least two detection elements configurable to measure an intensity and the method comprises further data processing methods during or after data acquisition that allow reconstructing the position of the signal from the intensities of parts of the signal measured by the detection elements.
Preferably, the detector according to this aspect of the invention comprises at least two detection elements configurable to measure a time of arrival, the method comprises further data processing methods during or after data acquisition that allow reconstructing the time of arrival of the signal from the times detected at the detection elements.
According to these last aspects, the evaluation of the determination of the position and the time of arrival of the signal can be performed.
According to a second aspect of the present invention, there is provided an apparatus comprising a sample holder; time signal means for providing a signal pulse; ionization means configured to ionize atoms or molecules of a sample on the sample holder by an ionization pulse at a fixed time relative to the signal pulse; imaging means configured to extract the ions from the sample and to accelerate them towards a generation means, whereby the distribution of the ions on the sample is isomorphously imaged to the generation means, the generation means being configured to generate a signal from an impinging ion indicative of the position of the impingement of the ion onto the signal generator; detector comprising two laterally separated detection elements configurable to detect if a part of said signal with at least a predefined intensity is received by said detection element; wherein at least one detection element is configurable to measure a time of arrival relative to the start time when said part of said signal with at least a pre-defined intensity is received by said detection element.
The apparatus according to the second aspect can be an apparatus for imaging mass spectroscopy.
More specifically, there is provided an apparatus comprising a sample holder; pulse generator for providing a signal pulse; a generator of an ionization configured to ionize atoms or molecules of a sample on the sample holder by an ionization pulse at a fixed time relative to the signal pulse; anode arrangement and imaging device configured to extract the ions from the sample and to accelerate them towards a signal generator, whereby the distribution of the ions on the sample is isomorphously imaged to the signal generator, the signal generator being configured to generate a signal from an impinging ion indicative of the position of the impingement of the ion onto the signal generator; detector comprising two laterally separated detection elements configurable to detect if a part of said signal with at least a predefined intensity is received by said detection element; wherein at least one detection element is configurable to measure a time of arrival relative to the start time when said part of said signal with at least a pre-defined intensity is received by said detection element.
According to the second aspect, such an apparatus is adapted to perform a method according to the first aspect of the invention.
Preferably, the detector of the second aspect of the invention comprises a detection element configured to measure an intensity of the received part of the signal.
More preferably, a detection element according to the second aspect of the invention is configurable to measure an intensity of the received part of the signal that is further configurable to measure the time of arrival of the received part of the same signal, or each detection element of a detector according to the second aspect of the invention is configurable to measure an intensity of said part of the signal and to measure the time of arrival of the received part of the same signal.
This last preferred aspect would allow for the highest spatial and time resolution possible with a given number of pixels per area.
Still preferably, the apparatus according to the second aspect of the invention comprises storage means configured to store the time of arrival of the part of the signal, the measured intensity of the received part of the signal and an identifier for each detection element as a data set; time reconstruction means configured for reconstructing the time of arrival of the signal from the stored times of arrival of the parts of the signal; and position reconstruction means configured for reconstructing the position of the impact of the signal from the measured intensities and the identifiers.
More specifically, the apparatus according to the second aspect of the invention comprises a detector and a memory configured to store the time of arrival of the part of the signal, the measured intensity of the received part of the signal and an identifier for each detection element as a data set; a time reconstructor configured for reconstructing the time of arrival of the signal from the stored times of arrival of the parts of the signal; and a position reconstructor configured for reconstructing the position of the impact of the signal from the measured intensities and the identifiers.
In embodiments according to these preferred aspects, the received intensity and time of arrival of parts of the signal may be used to determine the position and time of arrival of the signal more precisely than with other configurations of detection elements measuring a time and an intensity for an element.
Preferably, the detector according to the second aspect of the invention comprises a self-repetitive mosaic of detection elements spatially arranged in a repeating pattern such that detection elements configured to measure a time of arrival alternate according to a predefined rule with detection elements configured to measure an intensity.
E.g., the detector may comprise at least four detection elements arranged in an array of rows and columns and within each row detection elements configured to measure a time of arrival alternate with detection elements configured to measure an intensity; and within each column detection elements configured to measure a time of arrival alternate with detection elements configured to measure an intensity.
In embodiments according to this preferred aspect, a high spatial resolution of the measurement is combined with a high resolution of the elapsed time.
Preferably, the generation means according to the second aspect of the invention is a microchannel plate, the signal comprises electrons generated by the microchannel plate and the received intensity corresponds to the number of electrons impinging the detection element.
Preferably, the detector according to the second aspect of the invention is a semiconductor detector and the detection elements are pixel on the detector.
Still preferably, the apparatus according to the second aspect of the invention comprises at least two detectors, where the detectors are located adjacent to each other in the same plane, and where the pitch between adjacent detection elements on the same detector is substantially the same as the pitch between adjacent detection elements on adjacent detectors.
Thus, a detector system with a large area may be built. E.g. the pitch between adjacent detection elements on adjacent detectors may be not more than two to four times the pitch of adjacent detection elements on the same detector.
According to a third aspect of the invention, there is provided a computer program product embodied on a computer-readable medium, comprising program instructions which perform, when run on a computer, the execution of which result in operations of the data processing methods according to the first aspect of the invention.
Preferably, the computer program product according to the third aspect of the invention further comprises program instructions which perform, when run on a computer evaluating the number of signals with substantially the same lateral position and the same time of arrival when performing any of the data processing methods according to the first aspect of the invention and storing the evaluated number, the lateral position and the time of arrival in a set of data.
Other objects, features, and advantages of the present invention will become more apparent from the following detailed description of certain embodiments when read in conjunction with the accompanying drawings.
Herein below, certain embodiments of the present invention are described in detail, wherein the features of the embodiments can be freely combined with each other unless otherwise described. However, it is to be expressly understood that the description of certain embodiments is given for by way of example only, and that it is by no way intended to be understood as limiting the invention to the disclosed details.
With respect to
The imaging mass spectrometer as shown in
(1) The ionization chamber comprises a sample holder on an adjustable stage in order to position the sample. Ionization may be achieved by a pulsed LASER source of energetic photons of high flux that illuminates the extended surface of the specimen simultaneously. Depending on the molecules to be investigated, e.g. a N2 laser may be used, but other laser types are applicable, too. The intensity of the laser beam is regulated in an intensity regulator that generates an electronic pulse to set a start time of a measurement.
(2) The ions are separated by a time of flight mass separator. There, the ions are accelerated by an electrical field. The obtained speed depends on the m/z ratio of the ions, where m designates the mass and z the charge of the ion. The ions enter a drift region, where ions of different m/z-ratios are separated in time because of their different speed. The ions are accelerated and drift in the drift region towards the microchannel plate MCP. The distribution of the ions on the sample is isomorphously imaged onto the MCP. This may be achieved e.g. by a pinhole in the drift space. Alternatively, one or several lenses may be included to increase the angular acceptance and to sharpen the image. Thus, within the apparatus, several different lenses and combinations of these in different orders may be included. The drift part may be straight or bended by applying appropriate lenses or deflection elements. The bending angle may be between 0° and 180° depending on the configuration of lenses and deflection elements.
(3) The detector used in the embodiment together with the MCP is shown in more detail in
The detector comprises an array of pixels and generates a two-dimensional picture for a range of m/z values, i.e. a range of times of arrival, and their intensities simultaneously. It localizes ions fast and precise in two dimensions with its detector surface comprising pixels. The detector may be tuned for different spatial amplifications of the original specimen, sometimes also referred to as enlargement factor. The detector allows resolving the localization of molecules within the range of one to several square micrometers. The detector according to the present embodiment determines the time of flight of ionized molecules per each pixel and each measuring cycle simultaneously. The full mass spectrum in one pixel is reconstructed after a series of repeated measurements.
According to certain embodiments of the present invention, the time needed for the image acquisition is dramatically reduced compared to conventional methods. With the detector for mass spectroscopy according to certain embodiments, it is faster to acquire the full m/z spectrum and the location of the ions on the sample surface at the same time than to scan the sample surface point-wise. The acquisition of the m/z values for a large region of an extended surface is achieved simultaneously. Therefore, the time needed for e.g. bio-molecule discovery or acquisition of a molecular fingerprint is reduced significantly by the usage of the described detector.
Since the Time of Flight is measured for each ion, the imaging mass spectrometer can cover a large range of different m/z values. E.g. in bio-medical applications, the time of flight mass analyzer allows to detect small molecules and metabolites as well as alterations in protein composition within a given tissue, cell or even sub-cellular compartment.
In the detector, at pixel positions (x,y), the intensity of the signal In(x,y) or its Time of Arrival ToAn(x,y) are detected, step S104. The Time of Arrival is measured relative to the start time. There may be pixels arranged to measure intensity and pixels arranged to measure a Time of Arrival. The values In(x,y) and ToAn(x,y), respectively, are stored for each pixel (x,y), step 105. If enough measurements have not been performed yet to obtain a sufficient statistics, it is decided in S106, to return to S101. Otherwise, the measurement part of a duty cycle is finished.
In step S107, per measurement cycle n a position of one or several impinging electron clouds corresponding to an impingement position of an ion (x′,y′)n may be determined based on the intensities In(x,y) and a Time of Flight ToFn out of the Times of Arrival ToAn(x,y). The relationship between the positions (x,y) on the detector and the locations (X,Y) on the sample and between Time of Flight and Time of Arrival may be predetermined by calibration experiments. Thus, the location of an ion on the sample may be determined from the intensities In(x,y) on the detector. In step S108, a 4-dimensional image may be reconstructed from all cycles. For that, the data may be arranged in sets of substantially the same Time of Flight [ToF], representing a certain m/z ratio, substantially the same location ([X],[Y]) on the sample, and the number of events for this triplet. Finally, in step S109 the data may be evaluated and presented. The duty cycle is finished in silo.
In the following, the construction principle of an imaging mass spectrometer is described in detail including (1) a principle of surface ionization and ion ablation for (2) a time of flight detection with imaging capabilities, the principle of (3) ion detection and (4) image reconstruction, and its application to surface analysis techniques (5).
The kind of detector used according to certain embodiments of the present invention will influence the principle of the probe ionization process. In order to make use of the independent registration of ions produced at different spots on the sample surface, ionization over a large specimen area at the same time is achieved.
Molecules or atoms are ionized from a two-dimensional surface of interest at different spots at once. The ionization typically results in a few ions per pixel such that finally only one ion per pixel will be detected.
Certain embodiments of the present invention are applicable to a range of different molecules like bio-molecules including metabolites, proteins, lipids and single atoms. Either an ion beam or a LASER beam achieves the ionization of molecules accompanied by their transfer in the gaseous phase. In general, any technique that allows the ionization molecules to be present on an extended surface area is applicable. According to a certain embodiment of the present invention an ionization method is used that generates ions from a relatively large area of spots and not only from a single spot on the sample surface (e.g. only one molecule per ionization process and pixel).
Preferably, the ionization method deployed generates randomly ionized molecules of different m/z values from the molecules under investigation, e.g. bio-molecules. The summary of all ionized molecules detected subsequently reflects then a broad spectrum of molecules present on the sample surface. The ionization process according to certain embodiments is designed in such that it ionizes with almost no selectivity for a specific molecule under investigation and in contrast allows the ionization of any molecule. Any technique that ionizes molecules from a large area (μm to cm) with very low lateral diffusion of the ions subsequent to the ionization process may be used to achieve high spatial resolution. Advantages of the detection method according to certain embodiments are that (1) the ionization process is relatively non-selective with respect to the ionized molecule under investigation and (2) allows the generation of different m/z ions at several spots on the sample, and that (3) it generates only one ion or a small number during each ionization cycle (preferably not more than one ion per detector pixel) so that the detector is not saturated with incoming ion signals that would restrict the resolving capacity of the detector.
In order to achieve this ionization, a de-focused LASER beam may be used to illuminate the area of interest like in a typical NIMS experiment (Nanostructure Initiator Mass Spectrometry). As only one ion per detector pixel will be generated, a weak ionization procedure might be advantageous.
In certain embodiments, the ionization intensity is controlled by an intensity regulator in the path of the laser light. It may be used for generating a pulse for resetting the timers allocated to the pixels of the detector. Alternatively, e.g. a trigger signal for generation of the laser pulse or a signal of a light detector receiving light from the laser may be used.
The speed and success of the imaging mass spectrometer according to certain embodiments of the present invention is based on the fact that it is easier to achieve saturation in each mass spectrum and pixel of the detector than to scan the whole surface spot by spot as according to conventional scanning methods. E.g. for certain applications 50.000 to 100.000 individual ions of identical or different m/z values measured may be enough to generate a significant mass spectrum per pixel. Apparently, it is very efficient to measure a single ion per pixel at the same time across the area of interest. The sampling procedure may be stopped, once enough events for an m/z value are collected and the number of m/z values measured is diverse enough to provide enough information.
According to certain embodiments of the invention it is possible to reconstruct correctly the location of the ions generated at the surface of a sample, e.g. a tissue section. The principle of the time of flight mass spectrometer of certain embodiments is the selection and focussing of the ion bundle in the time of flight sector of the instrument such that it maps the ions generated at the surface of the sample on the detector surface only with minimal two dimensional distortion (e.g. caused by a low transversal momentum component).
In certain embodiments, the drift part of the imaging mass spectrometer is constructed in such that it corrects during the time of flight for m/z molecules with different initial kinetic energies. The drift element may let pass ions of a broad mass range and corrects for their spatial distortion resulting from the different initial kinetic energies acquired by the ions during the ionization process. For that purpose, a simple pinhole, electrostatic lenses or electrostatic field sectors may be used. This may also be done by the known method of delayed extraction, by use of a reflectron or in a switched time of flight mass spectrometer.
In some applications it might be necessary to select for a specific m/z range. In certain embodiments a narrow m/z distribution of ions may be selected to let pass. E.g. in case multiple ions are generated at the same spot of the pixel but of different m/z value, ions of a certain m/z window may be selected or ions with an unwanted m/z may be excluded with blankers. As another example, the strong ionization of the matrix due to the MALDI approach produces a number of low mass ions that also enter the mass spectrometer and may arrive earlier than the signal of interest. In addition, such a restriction of the m/z range allows optimal focussing for the distortion correction within the drift part of the time of flight tube of the instrument.
Therefore, the imaging TOF instrumentation can also implement for example two electrostatic field shutters that allow a restriction of the m/z range analyzed to a specific, narrow bandwidth of m/z values. Additional electrostatic focussing ion lenses may be included. These focussing lenses may also be used to magnify the area of interest on the surface of the sample in order to allow a detailed image, for example a sub-cellular analysis of a tissue sample. In contrast to the conventional scanning principle for image reconstruction, certain embodiments of the present invention employ electrostatic lenses in order to visualize the surface of interest with stigmatic lenses.
The ions leaving the drift field of the mass spectrometer are post-accelerated by an electrostatic field, before hitting the surface of a microchannel plate (MCP). When hitting the MCP, post-amplified ions liberate one or multiple electrons that are multiplied in individual channels of the MCP. Typical signal amplifications achieved are in the magnitude of 103 to 107.
This amplified electron cloud is detected by an application specific integrated circuit (ASIC) semiconductor array chip positioned behind the microchannel plate. Typically, the electron cloud generated at the MCP hits several pixels of the chip.
The detector set up of certain embodiments allows recording the separation of ionized molecules according to their time of flight. In certain embodiments one or several ions per each cycle and pixel are detected. The time of arrival of electrons generated from a single ion or multiple ions is registered by specifically configured pixels on the detector surface. The time of arrival is measured relative to the reset pulse generated by the intensity regulator at the time of the laser beam, as outlined above.
In certain embodiments, the “TIMEPIX” detector of the MEDIPIX collaboration can be used (see Xavier Llopart Cudie, PhD thesis, Mid Sweden University, Sundsval, Sweden, 2007). Each pixel of the array chip may be operated in one of three modes, i.e. arrival time, time over threshold and event counting. In a so-called “mixed mode”, it is possible to configure some of the pixels to measure the time of arrival while other pixels measure simultaneously the intensity of the signal. The differently configured pixels may be located adjacent to each other, such that a good spatial resolution of the signal may be obtained.
The semiconductor chip array detector “TIMEPIX” is a highly integrated monolithic device (ASIC) of few square centimetres in size (array detector), typically between 1 cm2 to 6 cm2. Every detector pixel is of square size comprising a lateral length between 10 to 70 μm. The pixel sensitivity is extremely high and works noise free due to an individual pixel calibration. Each pixel has a front-end digitisation with respect to the time measurement. The time stamp recorded is stored in an individual register for each pixel. The accuracy of the spatial position is in the order of one to several micrometers depending of the pixel size and the accuracy of the time measuring clock. Typically, the precision of the clock is in the range of few nanoseconds using a special reconstruction code in a software program. The fast recording speed is accomplished by the data taking method. Each register associated with one pixel is read out in parallel in a fraction of a millisecond.
The semiconductor detector chips which can be used for certain embodiments of the present invention can either be used in a stand alone version or assembled to large detector areas. Thereby, larger detection surfaces without distortion or disruption are achieved. The acquisition of mass spectra can be further improved with a multi-hit capability per each pixel.
The high sensitivity and dynamic range provided by the ASIC array detector used in certain embodiments can be advantageous for the field of mass spectrometry.
The ASIC detector set up allows registering the arrival time of an event which thereby determines the m/z ratio of a detected ion. According to certain embodiments, the detector chip is connected to an external clock. Alternatively, a clock could also be integrated with the chip or may be provided for each pixel separately. The precise determination of the arrival time is done based on the electron-induced signal intensity. The finite rise time of the circuit may cause a small time shift. Therefore the array detector may be used in a checkerboard configuration such that adjacent pixels determine either the time of arrival or the number of electrons. This allows for correcting the time shift of the arrival time produced by the finite rise time of the circuit.
Certain embodiments of the present invention include the detection of the signal amplitude and the time of arrival at the detector in a “mixed” mode. It allows the reconstruction of the time of flight for each single ion that arrives at the detector pixel. Thereby the mass spectrometer realizes two aspects: A high spatial resolution, and a determination of the m/z ratio for one single ion that arrives at a single detector pixel. In addition, the time of flight mass spectrometer may be equipped with focusing electrostatic ion lenses such that the mass separator can be run under magnifying microscope conditions.
The recording time window of the ASIC detector and the fast readout of the detector array provide additional time savings in each measurement cycle and therefore increase sensitivity and/or speed. Especially, the noise suppression in each individual ASIC detector pixel is excellently suited to generate only data from events observed.
To acquire a m/z spectrum at a given location the ionization of the probe can be reduced such that multiple ionizations per pixel are avoided. In turn the image can be read out with the ASIC detector which allows the coverage of a large two dimensional area.
As an alternative to the TIMEPIX detector, e.g. the Gossip detector (V. M. Carballo et al.: The charge signal distribution of the gaseous micropattern detector gossip, RESMDD06 conference, Florence, Oct. 10, 2006, http://www.nikhef.n1/˜i56/Hartjes_Gossip—10-10-06-1.ppt) or some other position sensitive detector allowing for time resolution may be used. Moreover the gossip detector has the advantage that each pixel may be configured to measure intensity and time simultaneously.
From the single individual measurements, a four-dimensional picture (two lateral dimensions [X,Y], one m/z dimension [m/z], and the number of events) may be reconstructed. The number of events corresponds to the abundance of an ion with that m/z ratio at the location [X,Y] on the surface of the sample.
A software algorithm may be programmed such that it allows first the determination of the precise arrival time and the intensity of the signal. Basically, a cluster analysis is performed that determines the position of the signal, the intensity of the signal, and its arrival time. Following to a cluster analysis, the reconstruction of a four dimensional image will be performed. It is based on the following construction principle: First all data generated are written in one virtual space of four dimensions (two lateral dimensions [X,Y], one m/z dimension [m/z] derived from the arrival time, and the number of events).
The method according to certain embodiments is suitable for a fast image acquisition in order to determine e.g. a tissue fingerprint rapidly and reliably. The intensities and different masses recorded in each spectrum are therefore focused on the most important ones. Several pictures can be taken of the same specimen in a short series of time and the detected ions are resolved according to their time of flight and relative location. In total a number of about 10 to 10.000 individual ions per pixel may be measured simultaneously. Subsequently, it is possible to reconstruct the spectrum of the whole sample. This will allow reconstructing the spectrum in varying depth and precision depending on the question asked.
The following experimental setup shows the proof of principle of certain embodiments.
A time of flight mass spectrometer as shown in
Following the drift space, the post-acceleration, and the MCP the array detector are included in the vacuum chamber. The imaging mass spectrometer is connected to a FPGA based controller board which is interfaced to a PC.
Several different calibrants were used to proof the working principle of the detector.
Following a post-acceleration, ions hit the surface of a microchannel plate which amplifies the signal over several magnitudes. Typical amplifications of the signal can be achieved between 103 and 107.
Following the microchannel plate, the expelled electron cloud is detected by the two dimensional detector ASIC as described, which is positioned in a small distance from the microchannel plate, typically between 1.0 and 2.0 mm. This distance determines the diameter of the ion cloud that arrives at the ASIC detector. The detector localizes the arrival time of the electron cloud generated from different ions and their two dimensional distribution. Both, the time of Arrival, the intensity of the signal and the position are recorded at the same time. For that purpose, the pixelated area of the detector is subdivided in two principally different kinds of signal detection. One part of the detector records the arrival time of the signal whereas the second determines the signal intensity. Both types of detecting elements, the time resolving and the charge recording pixels, are arranged in a checkerboard fashion covering the sensitive surface of the detector chip. Distributions alternative to the checkerboard set up for the time and intensity resolving pixels are also possible.
To measure the Time of Arrival (ToA), a counter of each pixel arranged to record the time starts counting clock pulses when the pixel receives at least predetermined signal intensity. The counter is stopped at a fixed stop time relative to the start time. The stop time is determined such that all ions may reach the detector within this time. The number of counted clock pulses is thus a measure of the Time of Arrival, where earlier Times of Arrival correspond to larger number of counted clock pulses and vice versa.
The ASIC detector principle allows resolving the arrival time and the intensity of particles at the detector surface. A presentation resulting from a detector signal of a single measurement is shown in
Four different clusters can easily be distinguished whereof two clusters have a rather high intensity while the other ones have a medium and small intensity. The different intensity may be caused by impact of several ions at the same place or by different sensitivities of the MCP and/or the detector at different positions thereof. This last effect can be estimated from calibration measurements.
The Time of Arrival is evenly distributed within each cluster. In the present experiment, all ions are Fe+ ions, therefore all clusters have about the same time of arrival. Based on these data, a precise determination of the impact position and the Time of Arrival for each ion can be obtained.
As calibration substance, CsI was ablated by MALDI from a sample plate. In
Closer inspection of the correlation plot for the Cs+ signal shows that the arrival time measured is not completely independent of the total intensity. A “walk” of the signal to earlier arrival times at higher total intensities was observed. This “time walk” is compensated for in a post acquisition data treatment.
At low rates of ions, the Time of Arrival may additionally be read out with even higher precision from the MCP. Then, the times measured by the MCP would be correlated with the times measured by the detector.
To gain insight in the dispersion of the arrival time measurements, the number of observed events versus the arrival time is plotted in
The distribution of the arrival times of Cs+, Fe+, and 32S+ and the origin (0/0) were fitted by Gaussians and the positions produced a calibration curve as depicted in
In a second set of experiments, to show the spatial resolution of the detector, the lateral dispersion of the ions generated at the target plate was determined.
Images reconstructed from data acquired with mass spectrometers influence a wide range of different fields in research and applications. It has the potential to become a standard application in areas like material surface diagnostics and or diagnostics in medical care. In the following different fields of applications are described in which the imaging mass spectrometer disclosed here may be efficiently applied.
The imaging mass spectrometer disclosed here can be applied to the fields of material surface analysis being relevant for the imaging of the composition of surfaces. This improves the position sensitive diagnostics of deposited layers.
For example, during chip production, the surface of a chip is analyzed by imaging mass spectrometry in order to trace impurities during manufacturing.
Petrography and age dating through the isotopic distribution of the radioactive decay products can also be performed with the imaging mass spectrometer disclosed here. The imaging capability results in increase of sensitivity.
A technology based on mass spectrometric analysis of bio-molecules significantly impacts the understanding and cure of complex diseases like cancer. Indeed, mass spectrometric measurements allow the determination of the kind and number of bio-molecules, present in for example a cancer cell. This allows a distinction of these from adjacent cells. The mass spectrometer according to certain embodiments can be efficiently applied for high throughput mass spectrometric analyses. Therefore, it will have a major impact on how diagnostics of cancerous tissue will be performed in the future. Indeed, the application in imaging mass spectrometry in diagnostics allows for an unbiased identification of a tissue composition. When repeated measurements are performed and a large set of cell type specific bio-molecules are identified, a novel method to an analytic platform is set up that guides the discovery of new therapeutic interventions to complex diseases. For example, summing up all spectra obtained and all bio-molecules identified in a specific cell type allows reconstructing a bio-molecular profile or “molecular fingerprint” of this specific cell.
Molecular fingerprints can identify and characterize alterations and in cellular content like changed metabolic pathways in cancer cells or even distinguish normal and malignant cell types.
Mass spectrometers with imaging capabilities can visualize heterogeneous distributions of bio-molecules for example on a tissue section. On a tissue biopsy it can determine and spatially resolve the presence and location of aberrant cellular content, which can be a consequence of abnormal cell metabolism for example in cancer cells. Certain embodiments of the present invention may allow discriminating cells based on differences in their respective stereotyped molecular fingerprints fast and reliable. The imaging mass spectrometer according to certain embodiments can be applied to detect molecules in a tissue that are specific for one type of tissue only and thereby enable a distinction of two different kinds of tissue on the basis on the relative abundance and number of specific bio-molecules present in a sample. In contrast to traditional staining techniques utilized to differentiate between tumorigenic tissue and normal tissue, the development of an imaging mass spectrometer allows to determine in a relatively unbiased and fast way a number of different biomarker quantitatively and spatially. This is in large advantage to standard methods like for example immuno-histochemistry, a slow and rather inefficient method to stain tissue section for only one specific antigen. The fast processing of tissue section for imaging mass spectrometry makes this image acquisition an ideal technique to acquire and perform analytical routine inspections of biomedical samples. Due to its specific advantages the imaging mass spectrometric method according to certain embodiments allows to sample large datasets in a fraction of time in order to build a database of molecular fingerprints from different tissues. This biometric reference database can be accessed later in order to enable a fast an unrestricted identification of normal tissue cell, the origin of the cell and the origin of its tumour cells.
The present invention is not limited to the embodiments described, but variations and modifications may be made without departing from the scope of the invention as defined in the appended claims.
Described above are certain embodiments according to which a method of imaging mass spectroscopy and a corresponding apparatus are provided, wherein the m/z-ratio of ions as well as the location of said ions on a sample surface are detected simultaneously in a time of flight mass spectrometer. The detector is a semiconductor array detector comprising pixels, that each can be arranged to measure a signal intensity of a signal induced by the ions or their time of arrival. A four-dimensional image consisting of the two lateral dimensions on the sample surface, the m/z-ratio representing the ion type and the abundance of an ion type on the surface can be reconstructed from repeated measurements for which a correspondingly adapted computer program product can be involved.
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|U.S. Classification||250/287, 250/288|
|Cooperative Classification||H01J49/40, H01J49/025, H01J49/0004|
|European Classification||H01J49/00B, H01J49/40, H01J49/02B|